食欲素A通过p38丝裂原活化蛋白激酶途径加重转染了淀粉样前体蛋白瑞典突变体（APPswe）的SH-SY5Y细胞的细胞毒性和线粒体损伤。
Orexin-A aggravates cytotoxicity and mitochondrial impairment in SH-SY5Y cells transfected with APPswe via p38 MAPK pathway.
作者信息
Li Maoyu, Meng Yao, Chu Bingcong, Shen Yang, Liu Xiangtian, Ding Mao, Song Chaoyuan, Cao Xi, Wang Ping, Xu Linlin, Wang Yun, Xu Shunliang, Bi Jianzhong, Xie Zhaohong
机构信息
Department of Neurology, Second Hospital of Shandong University, Jinan 250033, China.
Department of Neurology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Beijing 100005, China.
出版信息
Ann Transl Med. 2020 Jan;8(1):5. doi: 10.21037/atm.2019.11.68.
BACKGROUND
Alzheimer's disease (AD) is one of the common neurodegenerative diseases and is characterized by the accumulation of amyloid-β (Aβ). Orexin-A is a neuropeptide produced in the hypothalamus and thought to be involved in the pathogenesis of AD. However, its underlying mechanism and signaling pathway remains unclear. The aim of this work was to investigate the effect of Orexin-A on AD, and to explore its potential mechanism and signaling pathway.
METHODS
SH-SY5Y cells that were stably transfected with the Swedish mutant amyloid precursor protein (APPswe), a cell model of AD with excessive Aβ production, were used in this study. Cells were treated with Orexin-A, and with or without SB203580, an inhibitor of the p38 mitogen-activated protein kinase (MAPK) pathway, one of the key MAPK pathways associated with cell death. Following treatment, cells were collected and analyzed by western blotting, ELISA, electron microscopy, real-time PCR, fluorescence microscopy, and other biochemical assays.
RESULTS
Orexin-A increased the level of Aβ and Aβ in the cell medium, and activated the p38 MAPK pathway. As evidenced by the CCK-8 and ELISA BrdU assays, Orexin-A decreased cell viability and cell proliferation. Electron microscopic analysis used to observe the morphology of mitochondria, showed that Orexin-A increased the percentage of abnormal mitochondria. Further, decreased activity of cytochrome c oxidase (CCO), level of ATP, and mitochondrial DNA (mtDNA) copy number following Orexin-A treatment showed that Orexin-A exacerbated mitochondrial dysfunction. The level of intracellular reactive oxygen species (ROS), which is mainly generated in mitochondria and reflects mitochondrial dysfunction, was also increased by Orexin-A. SB203580 blocked the cytotoxicity and mitochondrial impairment aggravated by Orexin-A.
CONCLUSIONS
These findings demonstrate that Orexin-A aggravates cytotoxicity and mitochondrial impairment in SH-SY5Y cells transfected with APPswe through the p38 MAPK pathway, and suggest that Orexin-A participates in the pathogenesis of AD, which may provide a new treatment target in the future.
背景
阿尔茨海默病（AD）是常见的神经退行性疾病之一，其特征为β淀粉样蛋白（Aβ）的积累。食欲素A是在下丘脑产生的一种神经肽，被认为参与了AD的发病机制。然而，其潜在机制和信号通路仍不清楚。本研究的目的是探讨食欲素A对AD的影响，并探究其潜在机制和信号通路。
方法
本研究使用稳定转染了瑞典突变淀粉样前体蛋白（APPswe）的SH-SY5Y细胞，该细胞模型可过量产生Aβ，是AD的细胞模型。细胞用食欲素A处理，同时使用或不使用SB203580，它是p38丝裂原活化蛋白激酶（MAPK）通路的抑制剂，p38 MAPK通路是与细胞死亡相关的关键MAPK通路之一。处理后，收集细胞并通过蛋白质印迹法、酶联免疫吸附测定（ELISA）、电子显微镜、实时聚合酶链反应（PCR）、荧光显微镜及其他生化检测方法进行分析。
结果
食欲素A增加了细胞培养基中Aβ的水平，并激活了p38 MAPK通路。CCK-8和ELISA BrdU检测表明，食欲素A降低了细胞活力和细胞增殖。用于观察线粒体形态的电子显微镜分析显示，食欲素A增加了异常线粒体的比例。此外，食欲素A处理后细胞色素c氧化酶（CCO）活性降低、三磷酸腺苷（ATP）水平降低以及线粒体DNA（mtDNA）拷贝数减少，表明食欲素A加剧了线粒体功能障碍。主要在线粒体中产生并反映线粒体功能障碍的细胞内活性氧（ROS）水平也因食欲素A而升高。SB203580可阻断食欲素A加重的细胞毒性和线粒体损伤。
结论
这些发现表明，食欲素A通过p38 MAPK通路加重了转染APPswe的SH-SY5Y细胞的细胞毒性和线粒体损伤，并提示食欲素A参与了AD的发病机制，这可能为未来提供一个新的治疗靶点。
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